Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Specific mutations in an amino acid sequence are represented herein as AXaa1nXaa2″ where Xaa1 is the original amino acid residue before mutation, n is the residue number and Xaa2 is the mutant amino acid. The abbreviation “Xaa” may be the three letter or single letter (i.e. “X”) code. The amino acid residues for Hepatitis B virus DNA polymerase are numbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD (SEQ ID NO:10)) being residue number 550.
Hepatitis B virus (HBV) can cause debilitating disease conditions and can lead to acute liver failure. HBV is a DNA virus which replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy (1). The HBV genome is of a complex nature having a partially double stranded DNA structure with overlapping open reading frames encoding surface, core, polymerase and X genes. The complex nature of the HBV genome is represented in FIG. 1.
The presence of an HBV DNA polymerase has led to the proposition that nucleoside analogues could act as effective anti-viral agents. Examples of nucleoside analogues currently being tested are penciclovir and its oral form famciclovir (2, 3, 4, 5), lamivudine (6,7). Adefovir has been shown to have effective anti-HBV activity in vitro. Generally, such nucleotide analogues are used in conjunction with Hepatitis B immunoglobulin (HBIG) therapy. There is potential for such agents to be used in the treatment of chronic HBV infection.
Penciclovir has been shown to have potent inhibitory activity against duck HBV DNA synthesis in vitro and has been shown to inhibit HBV DNA polymerase-reverse transcriptase activity in vitro (8,9). Similarly, oral famiciclovir has been demonstrated to inhibit intra-hepatic replication of duck HBV virus in vivo (10). In man, famciclovir has been shown to reduce HBV DNA replication in a patient with severe hepatitis B following orthotopic liver transplantation (OLT) (11).
In work leading up to the present invention, nucleoside analogue antiviral therapy was used to control severe post-OLT recurrence of HBV infection (12). Long term therapy is mandatory where patients are immunosuppressed and the rate of HBV replication is very high. However, under such conditions, as with any long term chemotherapy of infectious agents, there is a potential for development of resistance or reduced sensitivity to the therapeutic agents employed. In addition, some patients do not respond to famciclovir pre-OLT. This may be due to patients not metabolising famciclovir or patients infected with a famciclovir-resistant HBV variant.
In accordance with the present invention, the inventors have identified variants of HBV with mutations in the HBV DNA polymerase gene which to varying extents reduce the sensitivity of HBV to nucleoside analogues. The identification of these HBV variants is important for the development of assays to monitor nucleoside analogue therapeutic regimes and to screen for agents which can mask the effects of the mutation, i.e. in the development of new vaccines. In addition, since the HBV genome comprises a series of overlapping open reading frames, a nucleotide mutation in one open reading frame can affect translation products in another open reading frame. In further accordance with the present invention, the inventors have observed mutations which reduce the interactivity of immunological reagents, such as antibodies and immune cells, to viral surface components. Such viral variants are referred to herein as “escape mutants” since they potentially escape existing immunological memory.